Compound, WF002, production thereof and use thereof

ABSTRACT

The present invention provides a new bioactive compound of formula (I), which has an antimicrobial activity against pathogenic microorganisms, and a process for production thereof. Also provided are a pharmaceutical composition comprising the compound and pharmaceutically acceptable carrier, a use of the WF002 as a medicament and use of the compound for manufacture of the medicament for treatment of infectious disease.

TECHNICAL FIELD

This invention relates to a new antimicrobial compound. Moreparticularly, it relates to a new antimicrobial compound that has anantimicrobial activity against pathogenic microorganisms, especiallypathogenic fungi, to a process for the preparation thereof and to apharmaceutical composition comprising the same.

DISCLOSURE OF INVENTION

The new compound, WF002 has the following physicochemical properties:

a) Molecular weight: ESI-MS (negative): m/z 689 (M−H)⁻

b) Elemental analysis: C, 61.08; H, 8.83

c) Melting point: 210-211° C.

d) Optical rotation: [α]_(D) ²³=+58° (c=0.5, methanol)

e) UV absorption spectrum: λ max(ε)=260 nm (methanol, 13000)

f) IR absorption spectrum: ν max(KBr)=3430, 2950, 2880, 1710, 1620,1450, 1370, 1260, 1100, 1080, 1040 cm⁻¹

g) ¹H-NMR spectrum: (500 MHz, CD₃OD) δ(ppm): 5.11 (1H, m), 4.98 (1H, s),4.37 (1H, d, J=8 Hz), 4.34 (1H, m), 4.08 (1H, m), 3.86 (1H, m), 3.67(1H, m), 3.38 (1H, d, J=10 Hz), 3.32 (1H, m), 3.28-3.20 (3H, m), 3.13(1H, m), 2.58 (1H, m), 2.28 (1H, m), 2.19 (1H, m), 2.06 (3H, s),2.06-1.95 (3H, m), 1.81-1.50 (7H, m), 1.49-1.20 (4H, m), 1.22 (3H, d,J=8 Hz), 1.20 (3H, s), 1.13 (3H, s), 1.12 (1H, m), 0.98 (3H, s), 0.97(1H, m), 0.93 (3H, s), 0.92 (3H, s).

h) ¹³C-NMR spectrum: (125 MHz, CD₃OD) δ(ppm): 211.3 (s), 173.2 (s),157.3 (s), 136.9 (s), 105.9 (d), 95.2 (d), 89.3 (d), 78.3 (d), 77.8 (d),75.9 (d), 71.9 (d), 71.7 (d), 63.0 (t), 59.4 (t), 56.4 (d), 52.7 (d),45.94 (d), 45.90 (t), 45.2 (s), 44.9 (s), 44.4 (t), 44.1 (s), 42.1 (s),39.5 (s), 35.6 (t), 35.0 (t), 31.3 (d), 30.1 (t), 28.7 (q), 28.1 (q),24.9 (t), 23.0 (t), 21.7 (q), 21.1 (q), 19.4 (q), 19.2 (t), 18.3 (q),17.9 (q).

i) Solubility

Soluble: methanol, ethyl acetate, dimethylsulfoxide

Insoluble: water, n-hexane

j) Color reaction

Positive: reactions with iodine vapor and cerium sulfate, and Molisch'stest, respectively.

Negative: reactions with ninhydrin, Ehrlich's reagent, Dragendorffreagent and ferric chloride, respectively.

k) Nature of substance: Neutral substance

l) Thin layer chromatography:

Carrier: Silica gel 60 F254 (Merck)

Solvent: dichloromethane:methanol=8:1

Rf=0.21

From the above physicochemical properties and extensive studies, theprovisional chemical structure of WF002 was assigned as follows.

According to this invention, the compound, WF002 can be prepared byculturing a WF002-producing strain, especially belonging to the genusMyrothecium in a nutrient medium.

Particulars of microorganisms used for the production of WF002 andproduction thereof will be explained in the followings.

Microorganism

The microorganism which can be used for the production of WF002 is aWF002-producing strain belonging to the genus Myrothecium, among whichMyrothecium cinctum No.002 was newly isolated from a material of Japan.

Lyophilized samples of the newly isolated microorganism, the strainNo.002 were deposited with an International Depository Authority on theBudapest Treaty, National Institute of Bioscience and Human-Technology,1-3, Higashi 1-chome, Tsukuba-shi, Ibaraki-ken, 305-0046 Japan under thedeposit number FERM BP-6380 on May 26, 1998.

The fungal strain No.002 was originally isolated from a soil sample.This organism grew rather rapidly on various culture media, and formedorange white to yellowish white colonies. The strain produced manyconidial structures on the agar media, while it did not formedteleomorph. The conidial structures consisted of hyphal conidiophoresand phialidic conidia, and the conidial masses were dark green to darkgray. On Sabouraud dextrose agar, the strain formed sometimessporodochial conidiomata. Its mycological characteristics were asfollows.

Cultural characteristics on various agar media are summarized inTable 1. Culture on potato dextrose agar grew fairly rapidly, attaining3.5-4.5 cm in diameter two weeks later at 25° C. This colony surface wasplane to raised, felty to cottony, sulcate or wrinkly, exudate,sometimes sectoring, orange white to pale orange at the center and themargin, and gray at the middle. Many conidial structures were formed onthe media. The reverse color was pale orange to light orange. Colonieson corn meal agar grew restrictedly, attaining 1.5-2.5 cm in diameterunder the same conditions. The surface was plane, thin, sometimessectoring, white to orange white at the center and the margin, andbrownish gray to dark gray at the middle. The reverse was white toorange white. Many conidial structures were formed.

The morphological characteristics were mainly determined from thecultures on a Miura's LCA plate (Mura, K. and M. Kudo: Trans. Mycol.Soc. Japan, 11:116-118, 1970). The conidiophores were erect fromvegetative or aerial hyphae. They were semi-macronematous, hyaline,smooth to roughened, repeatedly branched, and formed a whorl of 2-4phialides at the tips. The phialides were discrete, acrogenous, hyaline,roughened to granulate, cylindrical, with differentiated collarettes,(9-)16-28(-34)×(1.5-)2-3 μm in size, and producing conidia in slimydrops. Conidia were enteroblastic, phialidic, subhyaline to dark green,oblique or longitudinal striate, one-celled, fusiform to lentiform, and8.5-12×2.5-3.5(-4.5) μm. Vegetative hyphae were smooth, septate, hyalineand branched. The hyphal cells were cylindrical and 1.5-5 μm in width.Chlamydospores were not observed. Sporodochia on Sabouraud dextrose agarwere composed of loosely aggregations of hyphae and conidiophores, and100-300 μm in diameter.

Strain No.002 was able to grow at the temperature range from 6 to 33°C., with the growth optimum at 19 to 22° C. These temperature data weredetermined on potato dextrose agar (made by NISSUI).

On the basis of comparing the morphological characteristics with fungaltaxonomic criteria by von Arx (J. A. von Arx: The Genera ofFungi—Sporulating in Pure Culture. 3rd ed., pp.315, J. Cramer, Vaduz.1974) and Barron (G. L. Barron: The Genera of Hyphomycetes from Soil.pp.364, Williams & Wilkins, Baltimore, 1968), strain No.002 wasconsidered to belong to the hyphomycete genus Myrothecium Tode (1790).Moreover, above characteristics were corresponded the speciesdescription of Myrothecium cinctum (Code) Sacc. (1886) by Domsch et al.(K. H. Domsch, W. Gams and T.-H. Anderson: Compendium of Soil Fungi.vol. 1, p.482, Academic Press, London, 1980), with few exceptions. Thus,we identified this isolate as one strain of Myrothecium cinctum, andnamed it Myrothecium cinctum No.002.

TABLE 1 Cultural characteristics of strain No.002. Media Culturalcharacteristics Malt extract agar* G: Rather restrictedly, 2.5-3.5 cm S:Irregular, plane, felty, formed some conidial structures, yellowishwhite (2A2) at the center, white to orange white (5A2) at the margin,and olive gray (1F2) at the middle R: Pale yellow (4A3) to grayishyellow (4B3), and olive brown (4E4) at the middle Potato dextrose G:Fairly rapidly, 3.5-4.5 cm agar (Difco 0013) S: Circular, plane toraised, felty to cottony, sulcate or wrinkly, exudate, sometimessectoring, formed many conidial structures, orange white (6A2) to paleorange (6A3) at the center and the margin, and gray (1E1) at the middleR: Pale orange (5A3-6A3) to light orange (5A4-6A4) Czapek's solution G:Rather restrictedly, 2.5-3.5 cm agar* S: Circular, centrally raised toumbonate, zonate, felty, radiately sulcate, exudate, formed few conidialstructures, and yellowish white (3A2) to orange white (6A2) R: Paleyellow (4A3) to pale orange (6A3) Sabouraud dextrose G: Rather rapidly,3.0-4.0 cm agar (Difco 0190) S: Circular to irregular, centrally raisedto umbonate, felty, radiately sulcate or wrinkly, exudate, grayishyellow (4B3) to orange white (6A2), and formed some grayish dots ofsporodochia R: Grayish orange (5B5) to brownish orange (5C4) Emerson YpSs agar G: Rather rapidly, 3.0-4.0 cm (Difco 0739) S: Circular, plane,felty, hygroscopic, formed many conidial structures, dark green(27F4-27F5), and orange white (6A2) at the margin R: Light orange(6A4-6A5) Corn meal agar G: Restrictedly, 1.5-2.5 cm (Difco 0386) S:Circular, plane, thin, sometimes sectoring, formed many conidialstructures, white to orange white (5A2) at the center and the margin,and brownish gray (5F2) to dark gray (1F1) at the middle R: White toorange white (5A2) MY20 agar* G: Fairly rapidly, 3.5-4.5 cm S: Circular,plane to centrally raised, felty to cottony, exudate, hygroscopic,abundantly formed conidial structures, greenish white (28A2) to greenishgray (28B2) at the center, white to orange white (6A2) at the margin,and dull green (28D4) to dark green (28F4) at the middle R: Olive brown(4E4-4F4), and yellowish white (4A2) at the margin Oatmeal agar G:Spreading broadly, 4.5-5.5 cm (Difco 0552) S: Circular, plane, felty,radiately sulcate, exudate, hygroscopic, sometimes sectoring, abundantlyformed conidial structures, dark gray (1F1) to dark green (27F4), andpale orange (6A3) at the margin Abbreviation G: growth, measuring colonysize in diameter, S: colony surface, R: reverse. *: The compositions ofmalt extract agar, Czapek's solution agar and MY20 agar were based onJCM Catalogue of Strains (Nakase, T., 6th ed., pp.617, Japan Collectionof Microorganisms, the Institute of Physical and Chemical Research,Saitama, 1995).

These characteristics were observed after 14 days of incubation at 25°C. The color descriptions were based on Methuen Handbook of Colour(Kornerup, A. and J. H. Wanscher, 3rd ed., pp.252, Methuen, London,1978).

It is to be understood that the production of the new compound, WF002 isnot limited to the use of the particular organism described herein,which is given for illustrative purpose only. This invention alsoincludes the use of any mutants which are capable of producing the WF002including natural mutants as well as artificial mutants which can beproduced from the described organism by conventional means, such asgenetic engineering, X-ray, ultraviolet radiation, treatment withN-methyl-N′-nitro-N-nitrosoguanidine and the like.

Production of WF002

The compound, WF002 can be prepared by culturing a WF002-producingstrain in a nutrient medium.

In general, WF002 can be produced by culturing the WF002-producingstrain in a nutrient medium containing assimilable sources of carbon andnitrogen, preferably under aerobic conditions (e.g. shaking culture,submerged culture, etc.).

The preferred sources of carbon are carbohydrates such as sucrose,glucose, glycerol, soluble starch and the like.

The preferred sources of nitrogen are cottonseed meal, soybean flour,yeast extract, peptone, gluten meal, corn steep liquor, dried yeast etc.as well as inorganic and organic nitrogen compounds such as ammoniumsalts (e.g. ammonium nitrate, ammonium sulfate, ammonium phosphate,etc.), urea, amino acid and the like.

The carbon and nitrogen sources need not be used in their pure form,because less pure material which contain traces of growth factors andconsiderable quantities of mineral nutrients, are also suitable for use.Further, there may be added to the medium mineral salts such as calciumcarbonate, sodium or potassium phosphate magnesium salts and the like.If the culture medium is foamed remarkably, a defoaming agent such asliquid paraffin, higher alcohol, plant oil, mineral oil and siliconesmay be added.

Preferred production conditions of WF002 in massive amount may include asubmerged aerobic cultural condition.

Preferred production conditions of WF002 in small amount may include ashaking or surface culture in flask or bottle.

In case where the production is carried out in a large tank, it ispreferable to use the vegetative form of the organism for inoculation inthe production tank in order to avoid growth lag.

Agitation and aeration of the culture broth may be accomplished in avariety of ways. Agitation are provided by a propeller or the similarmechanical agitation equipment, by revolving or shaking the fermentor,by various pumping equipment or by the passage of sterile air throughthe medium. The fermentation is usually conducted at a temperaturebetween 20° C. and 35° C., preferably about 25° C. for 50 to 100 hours,which may be varied depending on the fermentation condition and scale.

Thus produced WF002 can be recovered from the cultured broth byconventional means which are commonly used for the recovery of otherfermentation products such as antibiotics.

In general, most of the WF002 produced are found in the culture filtrateas well as in the cells of the cultured broth. The WF002 can be isolatedfrom the filtrate and the cells of the cultured broth in a conventionalmanner such as concentration under reduced pressure, lyophilization,extraction with a solvent, pH adjustment, treatment with a resin (e.g.anion or cation exchange resin, non-ionic adsorption resin), treatmentwith an adsorbent (e.g. activated charcoal, silicic acid, silica gel,cellulose, alumina), crystallization, recrystallization and the like.

The WF002 have a strong antimicrobial activity against pathogenicmicroorganisms, especially pathogenic fungi such as, Aspergillusfumigatus, Candida albicans and the like. Accordingly, the WF002 isuseful as an antimicrobial agent, especially antifungal agent which isused for the treatment of infectious diseases in human beings andanimals.

As examples for showing such pharmacological effects of WF002, somepharmacological test data are illustrated in the followings.

Test 1 (Antimicrobial Activity)

Antimicrobial activity of WF002 was determined by a serial brothdilution method using 96-well microtiter plate in 100 μl of yeastnitrogen base dextrose medium. The inoculum was adjusted to 1×10⁵ colonyforming units/ml. Candida albicans and Aspergillus fumigatus werecultured at 37° C. for 24 hours and Cryptococcus neoformans was culturedat 37° C. for 48 hours in 5% CO₂ incubator. After incubation, the growthinhibition of microorganism in each well was determined by microscopicobservation. The results were shown as MEC (minimum effectiveconcentration: μg/ml) value (Table 2).

TABLE 2 Antimicrobial activity of WF002. Microorganisms MEC (μg/ml)Candida albicans FP633 6.25 Aspergillus fumigatus FP1305 0.04Cryptococcus neoformans YC203 50

The present antimicrobial agent comprising the WF002 is useful as atherapeutic agent for infectious diseases in animals including humanbeings.

The antimicrobial composition can be used in the form of pharmaceuticalpreparation, for example, in solid, semisolid or liquid form, whichcontains the WF002 in admixture with a pharmaceutical organic orinorganic carrier or excipient suitable for external, topical, enteral,parenteral, intravenous, intramuscular, or intramucous applications. Theactive ingredient may be compounded, for example, with usual non-toxic,pharmaceutically acceptable carriers for tablets, pellets, capsules,suppositories, solutions, emulsions, suspensions, ointments and anyother form suitable for use. The pharmaceutically acceptable carriersare water, glucose, lactose, gum acacia, gelatin, mannitol, starchpaste, magnesium trisilicate, talc, corn starch, keratin, colloidalsilica, potato starch, urea and other carriers suitable for use inmanufacturing preparations and in addition, auxiliary, stabilizing,thickening and coloring agents and perfumes. The antimicrobialcompositions can also contain preserving or bacteriostatic agentsthereby keeping the active ingredient in the desired preparations stablein activity. The active object compound is contained in theantimicrobial composition in an amount sufficient to produce the desiredtherapeutic effect upon the bacterially infected process or condition.

For applying this composition to human patients, it is preferably toapply it in a form of intraveneous, intramuscular, oral or percutaneousadministration. While the dosage or therapeutically effective amount ofthe WF002 varies depending on the age, conditions of each individualpatient to be treated, the preferred daily dosage of the WF002 can beselected from the range of 0.1-1000 mg/kg of the patient.

The following Example is given for the purpose of illustrating thisinvention, but not limited thereto.

BEST MODE OF CARRYING OUT OF THE INVENTION Example

(1) Fermentation of the Strain No.002 for the Production of WF002

An aqueous seed medium (160 ml) containing sucrose 2%, glycerol 2%,cottonseed meal 2%, dried yeast 1%, polypeptone 1%, KH₂PO₄ 0.1%, andTween 80 0.1% was placed in each of three 500-ml Erlenmeyer flasks andwas sterilized at 120° C. for 30 minutes. A loopful of the slant cultureof the strain No.002 was inoculated in each of the seed flasks. Theinoculated flasks were shaken on a rotary shaker (220 rpm, 5.1 cm throw)at 25° C. for 4 days, and 480 ml (three flasks) of the seed culture wereinoculated to 20 liters of sterile production medium consisting ofmodified starch 4%, glucose 1%, cottonseed meal 1%, gluten meal 0.6%,corn steep liquor 3%, (NH₄)₂SO₄ 1%, KH₂PO₄ 1.2%, Na₂HPO₄.12H₂O 1.9%,β-cyclodextrin 1%, Adekanol LG-109 (defoaming agent, Asahi Denka Co.,Ltd.) 0.05%, and Silicone KM-70 0.05% in a 30-liter jar fermentor.Fermentation was carried out at 25° C. for 4 days under aeration of 20liters/minute and agitation of 200 rpm.

The production of the WF002 in the fermentation broth was monitored byHPLC analysis indicated below.

# Analytical HPLC Condition

column: YMC Pack ODS-AM 303, S-5 120A (250×4.6 mm I.D., YMC Co., Ltd.)

mobile phase: 45% aqueous acetonitrile containing 0.5% NaH₂PO₄.2H₂O

flow rate: 1 ml/min.

detection: UV at 210 nm

retention time: 8.0 min.

(2) Isolation and Purification of the WF002

The culture broth (100 liters) was extracted with an equal volume ofacetone by stirring for 2 hours at room temperature. The mixture wasfiltered with an aid of diatomaceous earth. The filtrate was dilutedwith an equal volume of water and passed through a column (10 L) ofDIAION HP-20 (non-ionic adsorption resin, Mitsubishi Chemical Co., Ltd.)packed with 25% aqueous acetone. The column was washed with 50% aqueousmethanol (30 L) and then eluted with methanol (50 L). The activefraction (0-30 L) was diluted to 50 L with water and passed through acolumn (4 L) of YMC-GEL (ODS-AM 120-S50, YMC Co., Ltd.) packed with 50%aqueous methanol. The column was washed with 60% (12 L) and 70% (12 L)aqueous methanol and then eluted with 80% aqueous methanol (15 L). Theactive fraction (0-10 L) was diluted to 16 L with water and passedthrough a column (2 L) of YMC-GEL (ODS-AM 120-S50, YMC Co., Ltd.) packedwith 50% aqueous methanol. The column was washed with 70% aqueousmethanol (6 L) and eluted with 80% aqueous methanol (5.2 L). The activefraction (1-3.2 L) was concentrated under reduced pressure to dryness.The residue was dissolved in 50% aqueous methanol (2 L) and wassubjected to column chromatography on YMC-GEL (ODS-AM 120-S50, YMC Co.,Ltd. 2 L). The column was washed with 50% aqueous methanol (0.5 L) and30% aqueous acetonitrile containing 0.5% NaH₂PO₄.2H₂O (6 L) and elutedwith 40% aqueous acetonitrile containing 0.5% NaH₂PO₄.2H₂O (9 L). Theactive fraction (2-7 L) was diluted with an equal volume of water andpassed through a column (2 L) of YMC-GEL (ODS-AM 120-S50, YMC Co., Ltd.)packed with 20% aqueous acetonitrile containing 0.25% NaH₂PO₄.2H₂O. Thecolumn was eluted with 40% aqueous acetonitrile containing 0.5%NaH₂PO₄.2H₂O (9.7 L). The active fraction (4.7-7.7 L) was diluted withan equal volume of water and passed through a column (2 L) of YMC-GEL(ODS-AM 120-S50, YMC Co., Ltd.) packed with 20% aqueous acetonitrilecontaining 0.25% NaH₂PO₄.2H₂O. The column was washed with 50% aqueousmethanol (4 L) and 70% aqueous methanol (5 L) and eluted with 80%aqueous methanol (5.7 L). The active fraction (3-5.2 L) was concentratedin vacuo to give white precipitates. The precipitates were filtered anddried up to give 275 mg of the WF002 as white powder. This powder wasdissolved in a small volume of methanol and further purified bypreparative HPLC, using YMC-packed column (ODS-AM SH-343-5 AM S-5,250×20 mm I.D., YMC Co., Ltd.) with 45% aqueous acetonitrile containing0.5% NaH₂PO₄.2H₂O as a mobile phase and a flow rate of 9.9 ml/minute.Active fraction was diluted with an equal volume of water and passedthrough a column (2 L) of YMC-GEL (ODS-AM 120-S50, YMC Co., Ltd.) packedwith 22.5% aqueous acetonitrile containing 0.25% NaH₂PO₄.2H₂O. Thecolumn was washed with 40% aqueous methanol (4 L) and then eluted with75% aqueous methanol. The eluate was concentrated under reduced pressureto dryness. The residue was dissolved in a small volume of ethyl acetateand added with a large amount of n-hexane, and then was dried up to give197 mg of WF002 as white powder.

What is claimed is:
 1. A compound WF002 having the formula as follows:


2. A compound of claim 1 having the following physicochemical propertiesa) Molecular weight: ES1:MS (negative): m/z 689 (M−H) b) Elementalanalysis: C, 61.08; H, 8.83 c) Melting point: 210-211° C. d) Opticalrotation: [α]_(D) ²³=+58° (c=0.5, methanol) e) UV absorption spectrum: λmax(ξ)=260 nm (methanol, 13000) f) IR absorption spectrum: νmax(KBr)=3430, 2950, 2880, 1710, 1620, 1450, 1370, 1260, 1100, 1080,1040 cm⁻¹ g) ¹H-NMR spectrum: (500 MHZ, CD₃OD) δ(ppm): 5.11 (1H, m),4.98 (1H, s), 4.37 (1H, d, J=8 Hz), 4.34 (1H, m), 4.08 (1H, m), 3.86(1H, m), 3.67 (1H, m), 3.38 (1H, d, J=10 Hz), 3.32 (1H, m), 3.28-3.20(3H, m), 3.13 (1H, m), 2.58 (1H, m), 2.28 (1H, m), 2.19 (1H, m), 2.06(3H, s), 2.06 (3H, m), 1.81-1.5- (7H, m), 1.49-1.20 (4H, m), 1.22 (3H,d, J=8 Hz), 1.20 (3H, s), 1.13 (3H, s), 1.12 (1H, m), 0.98 (3H, s), 0.97(1H, m), 0.93 (3H, s), 0.92 (3H, s), h) ¹³C-NMR spectrum: (125 MHZ,CD₃OD) δ(ppm): 211.3 (s), 173.2 (s), 157.3 (s), 136.9 (s), 105.9 (s),95.2 (d), 95.2 (d), 89.3 (d), 78.3 (d), 77.8 (d), 75.9 (d), 71.9 (d),71.7 (d), 63.0 (t), 59.4 (t), 56.4 (d), 52.7 (d), 45.94 (d), 45.90 (t),45.2 (s), 44.9 (s), 44.4 (t), 44.1 (s), 42.1 (s), 39.5 (s), 35.6 (t),35.0 (t), 31.3 (d), 30.1 (t), 28.7 (q), 28.1 (a), 24.9 (t), 23.0 (t),21.7 (q), 21.1 (q), 19.4 (q), 19.2 (t), 18.3 (q), 17.9 (q), i)Solubility Soluble: methanol, ethyl acetate, dimethylsulfoxideInsoluble: water, n-hexane, j) Color reaction Positive: reactions withiodine vapor and cerium sulfate, and Molisch's test, respectively,Negative: reactions with ninhydrin, Ehrlich's reagent, Dragendorffreagent and ferric chloride, respectively.
 3. A process for thepreparation of the compound WF002 of claim 1 which comprises culturing acompound WF002-producing microorganism in a nutrient medium andrecovering the compound WF002 from the resultant cultured broth.
 4. Anantimicrobial agent comprising the compound of claim 1 and a carrier. 5.A pharmaceutical composition comprising an anti-microbial effectiveamount of the compound of claim 1 and a pharmaceutically acceptablecarrier.
 6. A method of killing microorganisms which comprises applyingthe compound of claim 1 to the microorganism.
 7. A method of killingfungi which comprises applying the compound of claim 1 to the fungi. 8.A method of treating an infectious disease caused by a pathogenicmicroorganism comprising applying the compound of claim 1 to saidpathogenic microorganism.
 9. An isolated culture of Myrothecium cinctumNo.
 002. 10. A process for the preparation of Myrothecium cinctum No.002 comprising culturing a Myrothecium cinctum No. 002-producingmicroorganism in a nutrient